Evaluation of HLA-DQ2/DQ8 genotype in patients with celiac disease hospitalised in 2012
at the Department of Paediatrics
Dorota A. Szałowska-Woźniak1, Leokadia Bąk-Romaniszyn2,3, Agnieszka Cywińska-Bernas1, Krzysztof Zeman1
1Department of Pediatrics, Preventive Cardiology and Clinical Immunology, Medical University of Lodz, Poland
2Department of Nutrition in Gastrointestinal Disease, Medical University of Lodz, Poland
3Department of Pediatrics, Immunology and Nephrology, Polish Mothers Memorial Hospital Research Institute, Lodz, Poland
Prz Gastroenterol 2014; 9 (1): 32–37 DOI: 10.5114/pg.2014.40848 Key words: celiac disease, antibodies, genotype, inheritance.
Address for correspondence: Dorota A. Szałowska-Woźniak MD, PhD, Medical University of Lodz, 4 Kościuszki St, 90-419 Lodz, Poland, phone:+48 606 28 28 06, e-mail: d.szalowska@interia.pl
Abstract
Introduction: Celiac disease (CD) is a permanent intolerance to gluten that occurs in genetically predisposed individuals and leads to small intestinal mucosa damage. According to ESPGHAN guidelines from 2012, CD can be diagnosed in a patient with characteristic clinical symptoms, in whom, anti-tissue transglutaminase antibodies (> 10 times the upper limit) are found, endomysial antibodies (EMA) is confirmed and a positive genetic test is obtained. In these conditions no small-bowel biopsies are required.
Aim: Evaluation of the presence of HLA-DQ2 and HLA-DQ8 haplotypes in children with previously diagnosed CD, hospitalised in 2012 at the Department of Paediatrics and Immunology and/or the Gastroenterological Outpatient Clinic, and their relatives.
Material and methods: Blood samples of 22 subjects, including 9 children with CD diagnosed on the basis of clinical symp- toms, serological investigations and small-intestine biopsy, 7 diagnosed on the basis of clinical symptoms and serological inves- tigations, 2 with the suspicion of CD on the basis of clinical symptoms and 4 relatives of a child with CD. Methods: HLA-DQ2/
DQ8 test, automatic evaluation by EUROArrayScan.
Results: The presence of HLA-DQ2 and/or HLA-DQ8 genotype was confirmed in 16 children with CD diagnosed on the ba- sis of clinical symptoms and serological tests with/without intestinal biopsy, in 2 with the suspicion of CD and in 1 relative of a celiac child.
Conclusions: The evaluation of HLA-DQ2/DQ8 haplotype confirms the genetic predisposition to CD in subjects with the dis- ease diagnosed previously on the basis of clinical symptoms, serological tests or intestinal biopsy. Genetic testing is particularly indicated for the diagnosis of CD in infants consuming gluten for a short time and in small amounts.
Introduction
Celiac disease (CD) is a genetically conditioned ab- normal response to gluten, which in the course of au- toimmune processes leads to small intestinal mucosa damage. Based on screening for CD performed in Europe and in the USA, its prevalence varies from 1 : 100 to 1 : 300 of the population, and for its symptomatic form – 1 : 3345 individuals [1]. However, despite the avail- ability of serological and genetic testing and intestinal biopsy, the ratio of individuals with diagnosed CD and non-diagnosed cases is 1 : 7, which is associated, among others, with different levels of physicians’ knowledge about this disease [1]. The prevalence of CD in children
and adolescents aged 2.5 to 15 years is 3–13/1000 of tested children, in Europe from 1 : 80 to 1 : 300 [2–4].
The results of the epidemiological investigations carried out in Poland in the years 2007–2009 indicated similar to the above for the incidence of celiac disease. It has been estimated that in Poland the number of children and adolescents up to 19 years of age suffering from CD was from 25,000 to as many as 75,000 individuals, and probably a significant amount remain undiagnosed [5]. Gluten is a fraction of proteins present in grains of wheat (gliadin), rye (secalin) and barley (hordein). Ex- posure to gluten, in genetically predisposed individuals, leads tothe development of immune reactions asso-
ciated with the activation of TCD4, CD8 lymphocytes, production of pro-inflammatory cytokines, excessive proliferation of intraepithelial lymphocytes, migration of macrophages to the intestinal wall and finally to intestinal mucosa damage (flattening of villi to total villous atrophy, crypt hypertrophy and the increase of intraepithelial lymphocytes) [6]. The severity of changes developing in small-bowel mucosa is classified by Marsh scale (1992) and modified by Oberhuber et al. [7–13].
In the course of celiac disease, besides the activation of T lymphocytes, there is induction of β lymphocytes, which produces specific antibodies – the markers for the disease. They include: tissue transglutaminase 2 (tTG2), endomysial antibodies (EMA) and the deamidated form of gliadin peptides (DPG). The determination of their concentration is routinely performed in IgA class, but in patients with serum immunoglobulin A deficiency (IgA
< 0.2 g/l) in IgG class [1, 14, 15]. HLA haplotype testing is of great value in the diagnosis of CD, and it also allows determination of the risk for this disease occurrence. In the ESPGHAN (European Society for Paediatric Gastro- enterology) guidelines from 2012, genetic testing has been included in the diagnostic management of celiac disease. It has been acknowledged that in the case of clinical symptoms of CD occurring in a patient togeth- er with the presence of anti-tissue transglutaminase antibodies (> 10 times the upper limit of normal), the presence of anti-endomysial antibodies and the pres- ence of class II HLA-DQ2 and/or DQ8 haplotype, CD can be diagnosed without the necessity of small-bowel biopsy [16]. HLA-DQ2 is present in 90–95% of CD pa- tients, whereas HLA-DQ8 is present in about 5–15% [17].
HLA-DQ2 and/or DQ8 allele is not found in only 1% of celiac patients. The presence of HLA-DQ2 and/or DQ8 is indispensable but insufficient for the development of the disease; HLA-DQ2 genotype is also observed in 25–30% and HLA-DQ8 in 5–10% of the general popu- lation [1]. Additionally, other genes not associated with MCH class II family (non-HLA genes), e.g. CTLA4 gene polymorphism (gene located on chromosome 2q33), PTP N22 (protein tyrosine-phosphatase nonreceptor 22), are responsible for susceptibility to CD [18–20]. HLA-DQ2 and HLA-DQ8 genes are located on chromosome 6p21.
HLA-DQ2 consists of HLA-DQA1*05 (subunit α) allele and HLA-DQB1*02 (subunit β) allele, and HLA-DQ8 gen- otype consists of HLA-DQA1*0301 (subunit α) allele and HLA-DQB1*0302 (subunit β) allele [21].
Aim
The aim of the study was to evaluate the presence of HLA-DQ2 and HLA-DQ8 haplotypes, predisposing to CD development in children with previously diagnosed celiac disease, hospitalised in 2012 at the Department
of Paediatrics and Immunology and/or the patients of the Gastroenterological Outpatient Clinic and their rel- atives.
Material and methods
The study included 22 subjects (13 women, 9 men):
20 children (aged from 11 months to 17.3 years) and 2 adults (father – aged 46.2 years and mother – aged 44 years, of a boy with CD), the mean age was in the whole group was 8.3 years. The investigated group comprised 9 children with CD diagnosed on the basis of characteristic clinical symptoms, serological investiga- tions and positive result of small-bowel biopsy, 7 children with the disease diagnosed on the basis of clinical symp- toms, serological investigations, without small-bowel bi- opsy, 2 children (11 and 14 months old) with suspected CD on the basis of clinical symptoms, and 4 first-degree relatives of a celiac child (mother, father, brother and sister) (Table I). In the group of 18 children 12 (67%) had coexisting diseases: 6 were treated due to allergy, 2 due to Duhring’s disease, 2 with bronchial asthma, 1 with diagnosed Crohn’s disease and hypothyroidism, 1 congenital optic nerve anomaly and reactive arthritis, 1 nephrotic syndrome and 1 girl was diagnosed with au- toimmune hepatitis.
Whole venous blood (2–3 ml) was used. The “EURO- ArrayScan” test was used for HLA-DQ2DQ8 determina- tion. The tests were performed in a laboratory experi- enced in performing such determinations. All patients and their parents were informed about the purpose of the determination and gave their written consent to perform their and their child’s genetic testing.
Results
In 19 (86.3%) of 22 individuals HLA-DQ2 or HLA- DQ8 was detected (12 in females and 7 in males).
Genetic predisposition to CD was confirmed in all 16 patients with CD diagnosed on the basis of clinical symptoms and positive results of serological investiga- tions with or without small-bowel biopsy, in 2 patients with suspected disease on the basis of clinical symp- toms (11- and 14-month-old children receiving gluten for a short time) and in 1 adult (mother of celiac boy), i.e. the presence of HLA-DQ2 and/or HLA-DQ8 genotype (Figures 1, 2). Among individuals with positive haplo- type associated with celiac disease, 13 (68%) had HLA- DQ2, 4 (21%) had HLA-DQ8, and 2 (11%) had HLA-DQ2 and HLA-DQ8 (Figure 3). In 3 individuals, first-degree relatives (father, aged 46 years, brother aged 12 years and 7 months, sister aged 19 months) of a boy with CD, no HLA-DQ2/DQ8 alleles were found. The boy’s brother and sister remained on a gluten-free diet due to sus- picion of CD despite negative results of serological in-
Table I. Characteristics of the investigated group
Children Patient’s
no:
Gender Age Clinical
symptoms of celiac disease
Biopsy Biopsy result
Coexisting diseases Serology results
1 M 10 years
8 months
Yes No – Congenital optic nerve anomaly,
reactive arthritis
tTGA positive, AGA negative
2 F 2 years Yes No – tTGA positive,
EMA positive, AGA positive
3 F 8 years
1 month
Yes Yes III c Inhaled allergy EMA positive
4 M 4 years
4 months
Yes No – Food and inhaled allergy,
bronchial asthma, atopic dermatitis
tTGA positive, EMA positive
5 M 16 years
3 months
Yes Yes IIIa Crohn disease, hypothyroidism tTGA negative, AGA positive
6 M 11 years
3 months
Yes Yes IIIb Bronchial asthma, polyvalent allergy tTGA positive, EMA positive
7 M 12 years
8 months
Yes Yes IIIc Nephrotic syndrome, polyvalent allergy tTGA positive, EMA positive
8 F 12 years
1 month
Yes Yes IIIc IgA nephropathy tTGA positive,
EMA positive
9 M 3 years
11 months
Yes No – tTGA positive,
AGA positive
10 M 1 year
2 months
Yes No – tTGA negative,
AGA negative
11 F 15 years
4 months
Yes Yes IIIc tTGA positive,
AGA positive
12 F 12 years
3 months
Yes Yes IIIb Duhring’s disease No before-diet
results
13 F 5 years
1 month
Yes Yes IIIc tTGA positive,
EMA positive
14 F 11 months Yes No – tTGA negative
15 F 1 year
2 months
Yes No – Duhring’s disease AGA positive
16 F 12 years Yes Yes IIIc tTGA positive
17 F 17 years
3 months
Yes No – Autoimmune hepatitis tTGA positive,
EMA positive
18 F 6 years
5 months
Yes No – tTGA positive
Celiac child first-degree relatives Siblings
19 F 1 year
9 months
No No – Food allergy tTGAnegative,
EMA negative
20 M 12 years
7 months
Yes No – Food allergy tTGA negative,
AGA negative Parents
21 M 46 years
2 months
Yes No – On gluten-free
diet:
tTGA negative, AGA negative
22 F 44 years No No – tTGA negative
vestigations but with traits of food allergy and irregular bowel movements. The father of the family eliminated gluten from his diet due to recurrent abdominal pain. In the mother, treated as healthy subject, after a positive result of genetic testing for celiac disease, a silent form of the disease was diagnosed.
Discussion
The genetic susceptibility to CD was confirmed in the tests of all children with intestinal mucosa atrophy (Marsh III), in serologically positive children, and even in two infants only with clinical symptoms of the dis- ease. In the Polish project of genetic testing of celiac disease, performed in the years 2007–2009, including 922 subjects: 594 with diagnosed celiac disease, 131 with suspicion for CD and 141 from celiac families, 79%
had a positive result of SNP genotyping (genetic varia-
tions of single nucleotide polymorphism) for DQ2 and/or DQ8. Positive results of genotyping were demonstrated in 97% of patients with diagnosed CD and in 47% of individuals suspected for CD [22]. In the study of Kipa- tany et al. in 2006 from the University in Debreczyn, similarly as in our study, all 40 investigated subjects, in whom the presence of anti-tissue transglutaminase antibodies and EMA was confirmed, had HLA-DQ2 or DQ8 allele [10]. The evaluation of the genotype for the presence of HLA-DQ2/DQ8, as we became con- vinced in our study, is a test allowing early recognition of CD in children who receive gluten for a short time and/or in a small amount. In the group investigated by us, a positive result of HLA-DQ2/DQ8 was obtained in a 14-month-old boy and positive HLA-DQ8 in an 18-month-old girl with insufficient weight gain and growth despite a negative result of serum tissue trans- Figure 2. Gender distribution in the population
of subjects up to 18 years old of the life with positive results of testing (n = 18)
Age interval
15–18
12–15
9–12
6–9
3–6
0–3
0 0.5 1.0 1.5 2.0 2.5 3.0 Number
♀ ♂
Figure 3. Distribution of HLA-DQ2/DQ8 haplo- types of subjects with positive result of genetic testing (n = 19)
14
12
10
8
6
4
2
0
HLA-DQ2 HLA-DQ8 HLA-DQ2/DQ8 Figure 1. Evaluation of HLA-DQ2/DQ8 genotype in the examined children (n = 20)
Siblings Clinical symptoms
Clinical symptoms, serology
Clinical symptoms, serology, biopsy
0 1 2 3 4 5 6 7 8 9 0
2
2
7
9 0
0
0
HLA-DQ2/DQ8– HLA-DQ2/DQ8+
glutaminase. In ESPGHAN guidelines low diagnostic reliability has been emphasized for serological investi- gations for CD in children < 2 years of age remaining on a gluten-free diet or consuming gluten for a short time and in a small amount, with accompanying diseases, in the evaluation of familial predisposition to the disease and in subjects treated with immunosuppressive agents [16]. It has been emphasised that a negative result of HLA-DQ2/DQ8 test excludes CD in 99% and a positive result points to the disease or to a predisposition to it [1, 16]. The performance of genetic testing is indicated to recognise or exclude CD in patients with ambiguous or contradictory results of serological and histopatho- logical investigations and also in individuals remaining on a gluten-free diet (when the results of histopatho- logical and serological tests can be false negative). The evaluation of HLA-DQ2/DQ8 is also indicated in the so- called risk groups, i.e. first-degree relatives of celiac pa- tients, patients with type I diabetes mellitus and pathol- ogies coexisting with CD (Down’s syndrome, Turner’s syndrome, Duhring’s disease and others). Confirmation of a predisposition to celiac disease by genetic testing is, for the patients, an additional confirmation of the va- lidity of the application of a restrictive gluten-free diet.
In our studies the negative result of genetic testing in 3 subjects allowed the exclusion of CD and enabled the decision to be made for gradual introduction of gluten into the diet, which allows the development of sensitiv- ity in an allergy. In the group investigated by us, in 67%
of children with celiac disease, chronic diseases were present including those of immunological background.
This is in compliance with the observations of other authors; CD often coexists with other diseases, most frequently with type I diabetes, autoimmune thyroiditis, IgA nephropathy, Down’s syndrome, Turner’s syndrome, Williams syndrome, isolated IgA deficiency and nonspe- cific intestinal diseases [23].
Conclusions
The evaluation of HLA-DQ2/DQ8 haplotype con- firms the genetic predisposition to CD in individuals with previously recognised disease on the basis of clin- ical symptoms, serological investigations or intestinal biopsy. A positive result of the genetic test confirming a predisposition to CD in a small child receiving gluten for a short time (or with clinical symptoms of the dis- ease) is an indication for further medical supervision and periodic serological testing.
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Received: 15.02.2013 Accepted: 15.09.2013
